Laboratory

Chromatography

Guide
Laboratory
Chromatography
Guide
Angelo Talamona
 Author Angelo Talamona
Publisher Büchi Labortechnik AG, CH-9230 Flawil, Switzerland
Cover NOSE Applied Intelligence AG, CH-8005 Zürich, Switzerland
Layout Atelier Güttinger AG, CH-9030 Abtwil, Switzerland

First edition All rights reserved. No part of this publication may be reprinted,
Printed in Switzerland or reproduced, or utilized in any form or by any electronic or
94175 0105 mechanical means – now known or hereafter invented –, including
photocopying and recording, or in any information storage
and retrieval system, without the publisher’s written permission.

ISBN 3-033-00339-7 © 2005 by Büchi Labortechnik AG, CH-9230 Flawil, Switzerland.

“Laboratory Chromatography Guide” – A close look at
preparative liquid chromatography

The present “Laboratory Chromatography Guide” is dedicated to

preparative liquid chromatography, a common purification techni-
que in most chemical or life science laboratories.
The performance of separations by chromatography is fairly
well known in the scientific and industrial communities. Part 1, the
“Flash Guide Basics”, gives consideration to this fact, proceeding
swiftly through flash chromatography with an emphasis on speed,
reliability and reproducibility of the separation.
But there are no rules without exceptions! As usual, problems
appear with the most exciting and valuable compounds you want
to purify. Therefore, you are personally challenged to understand
and solve the purification task as fast as possible. The second part
“Preparative Column Chromatography: Theory and Practice” helps
you to overcome such drawbacks and leads you back to the shining
path of your privileged profession: to understand and explore what
modern science offers!
We at Buchi, as a leading supplier of high quality laboratory pro-
ducts and responsive services, wish you a lot of challenging and
successful work!

Dr. Ernst Freydl

Büchi Labortechnik AG
 Contents

Part 1 Flash Guide

Basics

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2 Principle of chromatography . . . . . . . . . . . . . . . . . . . 14

3 Choice of the appropriate stationary phase . . . . . . . 15

4 Evaluation of the chromatographic system by

thin-layer chromatography . . . . . . . . . . . . . . . . . . . . . 16
4.1 Evaluation of the stationary phase . . . . . . . . . . . . . . . . . . . 16
4.2 Selectivity of the solvent . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.3 Solvent strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5 Injection/Column loading . . . . . . . . . . . . . . . . . . . . . . 23

6 Gradient elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Part 2 Preparative Column Chromatography
Theory and Practice

1 Starting point – Definition of the problem . . . . . . . . . 32

1.1 Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
1.2 Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
1.3 Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

2 Fundamentals – The basic principles . . . . . . . . . . . . . 34

2.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.2 Adsorption chromatography . . . . . . . . . . . . . . . . . . . . . . . 34
2.2.1 Separation mechanisms in adsorption chromatography . . . 34
2.3 Size exclusion chromatography . . . . . . . . . . . . . . . . . . . . . 36
2.4 Ion-exchange chromatography . . . . . . . . . . . . . . . . . . . . . 39
2.5 Affinity chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . 39

3 Stationary phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.2 Normal phase silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.3 Alumina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.4 Polyamides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.5 Reverse phase silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.6 Size exclusion chromatography . . . . . . . . . . . . . . . . . . . . . 44

4 Mobile phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4.2 Solvent strength and selectivity . . . . . . . . . . . . . . . . . . . . . 47
4.3 Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.4 Solvents for normal phase chromatography . . . . . . . . . . . . 49
4.5 Solvents for reversed phase chromatography . . . . . . . . . . 50
4.6 Solvents for gel chromatography . . . . . . . . . . . . . . . . . . . . 51

5 Deactivators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

6 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
6.1 UV detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
6.2 Refractive index detector . . . . . . . . . . . . . . . . . . . . . . . . . 55
6.3 Conductivity detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

7 Characterizing a column . . . . . . . . . . . . . . . . . . . . . . . 58
7.1 The chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
7.2 Symmetry index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
7.3 Number of theoretical plates . . . . . . . . . . . . . . . . . . . . . . . 60
7.4 Height equivalent to a theoretical plate . . . . . . . . . . . . . . . 61
7.5 Reduced plate height . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
7.6 Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
7.7 Dead volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
 Contents

8 Factors affecting chromatographic separation . . . . . 64

8.1 Capacity factor k’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
8.2 Separation factor α (selectivity factor) . . . . . . . . . . . . . . . . 67
8.3 Effect of α and k’ on the resolution . . . . . . . . . . . . . . . . . . 72
8.4 Effect of α and k’ on the number of theoretical plates N . . . 73
8.5 Effect of particle size on the column efficiency . . . . . . . . . . 74
8.6 Effect of flow rate on the column efficiency . . . . . . . . . . . . 76
8.7 Effect of column length on the number of theoretical plates . 77
8.8 Effect of column length on the resolution . . . . . . . . . . . . . . 77
8.9 Chromatography with several columns in series . . . . . . . . . 79
8.10 Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

9 Thin-layer chromatography as a pilot method

for column chromatography . . . . . . . . . . . . . . . . . . . . 81
9.1 Introduction to thin-layer chromatography . . . . . . . . . . . . . 81
9.2 Interpretation of TLC information . . . . . . . . . . . . . . . . . . . . 82
9.2.1 Calculation of the Rf value . . . . . . . . . . . . . . . . . . . . . . . . 82
9.2.2 Calculation of the separation factor α, capacity factor k’
and plate number N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
9.2.3 Resolution – Relationship of α and N to resolution . . . . . . . 84
9.3 Evaluation of stationary and mobile phase by means of TLC 85

10 Choice of the appropriate column . . . . . . . . . . . . . . . 88

11 Packing and conditioning of the column . . . . . . . . . . 89

11.1 General aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
11.2 Dry packing method for glass columns . . . . . . . . . . . . . . . 90
11.3 Packing method with Büchi Cartridger C-670 . . . . . . . . . . 92
11.4 Slurry packing method for silica . . . . . . . . . . . . . . . . . . . . 93
11.5 Packing method for soft and rigid gels . . . . . . . . . . . . . . . 94
11.6 Conditioning dry-packed columns . . . . . . . . . . . . . . . . . . . 96
11.7 Conditioning slurry-packed columns . . . . . . . . . . . . . . . . . 96
11.8 Conditioning gel columns . . . . . . . . . . . . . . . . . . . . . . . . . 96

12 Column test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
12.1 General aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
12.2 Test mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
12.2.1 Test mixtures for normal phase columns . . . . . . . . . . . . . . 98
12.2.2 Test mixtures for reversed phase columns . . . . . . . . . . . . . 100
12.2.3 Test mixtures for size exclusion gels . . . . . . . . . . . . . . . . . 101
12.2.4 Examples of test chromatograms . . . . . . . . . . . . . . . . . . . 101

13 Cleaning of columns . . . . . . . . . . . . . . . . . . . . . . . . . . 102

13.1 Cleaning of normal phase columns . . . . . . . . . . . . . . . . . . 102
13.2 Cleaning of reversed phase columns . . . . . . . . . . . . . . . . . 103
13.3 Cleaning of gel columns . . . . . . . . . . . . . . . . . . . . . . . . . . 103

14 Equipment description . . . . . . . . . . . . . . . . . . . . . . . . 104

15 Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
 Appendix

1 Common formulae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

2 Tables and graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
3 Solvent properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
4 Glossary, nomenclature and abbreviations . . . . . . . . . . . . . 120
5 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
6 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Abbreviations

TLC Thin-layer chromatography

HPLC High-performance liquid chromatography
[C]phase 1 Concentration of the compound C in phase 1
GC Gas chromatography
RPC Reversed phase chromatography
Si Solvent strength
RI Refractive index
S.I. Symmetry index
Fm Delivery rate
V0 Dead volume
GLP Good laboratory practice
MPLC Medium pressure liquid chromatography
LC Liquid chromatography
UV Ultraviolet
8

Introduction

Chromatography has developed very rapidly over the past few ye-
ars. It was a very long way from the first “capillary pictures” of
Runge (1822 –1850) through the early work of Tswett, the dis-
coverer of Adsorption Chromatography (1903, separation of plant
pigments) to modern HPLC from about 1967. Tswett had in fact
adopted the name “Chromatography” for this separation technique
(from the Greek chromos = colors, graphein = write).
However, the focal point of this enormous development was
clearly in the area of analysis. In preparative chemistry, on the
other hand, chromatographic separations are frequently carried
out even today by a very simple method, i.e. with the aid of a
simple glass column under hydrostatic pressure. The first publi-
cations on preparative chromatography under elevated pressure,
so-called Flash Chromatography, only appeared towards the end
of the seventies. This method too was subsequently further refi-
ned. This finally resulted in medium pressure liquid chromatography
(called MPLC in the following), which is very efficient but neverthe-
less readily comprehensible and simple to carry out. At the same
time, attempts were made to increase the size of the analytical
HPLC systems and thus make them available also for preparative
or at least semi-preparative work.
However, closer scrutiny reveals substantial differences between
routine analysis and preparative separation. It is therefore essential
for a preparative MPLC system to meet the specific requirements
for such separations. The following factors must be noted in parti-
cular:
– Flexibility in the choice of column. The amount of substance and
the required separating power differ for virtually every problem to
be solved. Simple and economical adaptation to the particular
separation problem must therefore be possible.
– High delivery of the pump. Large columns require large volume
flows so that the desired linear flow rate can be achieved.
– Wide pressure range. The trend in preparative chromatography
is clearly towards fine-grained adsorbents, which offer substan-
tial resistance to flow.
– The apparatus must be simple to handle. In particular, filling and
emptying of the columns as well as operation of the entire re-
maining system must be capable of being mastered immediately
without a prolonged familiarization period. In the preparative la-
boratory, the liquid chromatography is in general not a speciali-
zed unit but rather a universal tool.
 9

This booklet aims to provide both non-specialists and spe-

cialists with short and basic as well as with more detailed explana-
tions of the different procedure steps encountered during a liquid
chromatography separation.
The first part, “Quick Guide”, is a short, practice-oriented over-
view of liquid chromatography (LC) for quick reference searches
and the second part provides a broader and deeper description of
the process, under both practical and theoretical considerations.
Flash Guide
Basics

1
12 Part 1 Flash Guide – Basics

1 Introduction

Chromatography is a standard method used in preparative labora-

tories to isolate and purify substances. In the early days of chro-
matography simple glass columns were chiefly used, operated by
means of the hydrostatic pressure of the solvent acting as an elu-
ent. In a publication in 1978 Clark W. Still explored the possibility
of accelerating the separation process in simple glass columns,
which was until then the commonly used method, and thereby
considerably increasing the efficiency of the technique. The results
were convincing and the foundations of modern flash chromato-
graphy were laid. It triumphantly established itself in laboratories
as an indispensable purification method in preparative chemistry.
Flash chromatography has since undergone constant development,
and has been adapted to meet present day expectations in terms of
equipment and convenience.

Figure 1:
From the simple glass
column to modern flash
chromatography.

Modern flash chromatography systems are popular nowadays

because they are simple to handle, flexible and can be universally
employed. The first part of this brochure aims to give simple, acces-
sible advice, which should ideally instantly lead to effective labora-
tory elutions.
Introduction 13

The following abbreviations are used in the first part:

TLC Thin-layer chromatography

RP Reversed phase, modified silica gels
NP Normal phase polar silica gel phases
UV Ultraviolet
Si Solvent strength (substitutes polarity)
%A % solvent with low solvent strength
%B % solvent with high solvent strength
Rf Retention factor (from thin-layer chromatograms)
CV Column volumes
ΔCV Difference in column volumes
Rf1 Retention factor of first substance (substance which
spreads onto the TLC plate the quickest. The index
increases according to the time the substance takes to
spread).
14 Part 1 Flash Guide – Basics

2 Principle of chromatography

Chromatographic separation is based on a balanced state among

the components to be separated, an adsorbent agent in the column
(= stationary phase) and a solvent flowing through it (mobile phase).
When a component settles on the stationary phase this is defined
as adsorption, while detachment by the mobile phase is defined as
desorption. A high adsorption capacity between the components
of interest and the stationary phase means that there is a high
retention of these components and that there is a considerable
delay in elution from the column. The separation of a mixture into its
individual components is only possible if the individual components
in a combination of stationary and mobile phases have different
adsorption/desorption properties.

Figure 2:
Adsorption und
Desorption, schematic
illustration of the
chromatographic
separation process.
 15

3 Choice of the appropriate stationary phase

Chromatographic separation can be carried out on both polar and

apolar stationary phases, and suitable sorbents are available from
various manufacturers.
“Standard” chromatography requires the use of polar stationa-
ry phases such as silica gel and nonpolar solvents. The individual
components are delayed as a result of a reaction between the polar
function component groups and the polar groups of the sorbent.
Low polarity substances are eluted first, followed by components
of increasing size.
In “reversed phase” chromatography, however, the stationa-
ry phase is nonpolar and elution is by means of polar solvents.
These stationary phases are produced by modifying silica gel with
nonpolar groups such as C-18 or similar substances. Substances
are eluted in order of decreasing polarity from reversed phase co-
lumns, i.e. the substance with the highest polarity appears first.
Reversed phase materials are considerably more expensive than
standard stationary phases, and this is one of the reasons why
standard stationary phases are primarily used in flash chromato-
graphy. If the substance classes to be separated allow, modified
stationary phases can nonetheless be used without restrictions or
problems.

Figure 3:
Elution sequence for
normal silica gel.
16 Part 1 Flash Guide – Basics

4 Evaluation of the chromatographic system by

thin-layer chromatography (TLC)

As mentioned earlier, most elutions in flash chromatography use

normal silica gel, or modified silica gel in special cases or for highly
polar substances. In all these cases it is advisable to carry out a
thorough TLC pre-elution so that, with a minimum investment of
time and material, promising elution conditions can be found, which
can then be applied to the cartridge. The following applies:
1. Define stationary phase
2. Find mobile phase with best selectivity
3. Set solvent strength

Ideally the sorbents on the TLC plate and in the cartridge should
be identical (type and pore size) so as to successfully apply TLC
conditions to the cartridge!

4.1 Evaluation of the stationary phase

The laboratory’s experience with TLC tests, with which most la-
boratories are familiar, can help to you make the right choice. If
TLC plates with normal silica gel are used for the tests, column
separation can also be carried out using normal silica gel. If the
results of this prove unsatisfactory, it is then advisable to switch to
RP plates.

4.2 Selectivity of the solvent

Once the stationary phase has been established the mobile phase
with the most suitable selectivity needs to be found, i.e. the sol-
vent or solvent mixture that isolates the substance of interest on
Figure 4:
Selectivity triangle
with various selectivity
groups.
Evaluation of the chromatographic system by thin-layer chromatography (TLC) 17

the TLC plate with the greatest possible distance to the adjacent
components.
In general every solvent has its own defined selective proper-
ties; some tend to be similar to each other, while others can differ
greatly. L.R. Snyder and J.J. Kirkland investigated and compared
the properties resulting from various solvents and grouped solvents
with similar effects together into what are known as Selectivity
Groups.
The selectivity groups allow us to focus our search. There is
little point in comparing different solvents from the same selectivity
group, as they all have the same properties. What we have to do
is compare solvents from the various selectivity groups, as this is
the only way to see the difference immediately. The most important
solvents for our separation are compiled in the following table. This
only shows solvents that are suitable for separation with UV de-
tection, and do not make detection impossible as a result of high
energy absorption.
Solvent Group Strength Si UV limit Table 1:
The most common
n-Hexane – 0.1 200
solvents with selectivity
Cyclohexane – 0.2 210 group allocation and
solvent strength Si .
Diisopropyl ether I 2.4 220
Diethyl ether I 2.8 220
Ethanol II 4.3 200
Methanol II 5.1 200
Tetrahydrofuran III 4.0 220
Acetic acid IV 6.0
Dichloromethane V 3.1 250
Ethyl acetate VI 4.4 260
Aceton VI 5.1 330
Acetonitrile VI 5.8 210
Toluene VII 2.4 290
Xylene VII 2.5 290
Chloroform VIII 4.1 250

Depending on the polarity of the components to be separated,

the entire mixture can flow onto the TLC plate with the solvent
front; the solvent is too strong and the TLC separation cannot be
assessed in this form. In these cases the solvent strength is redu-
ced by diluting the solvent with hexane, for instance, and the TLC
separation is then repeated.
18 Part 1 Flash Guide – Basics

Figure 5:
Reducing solvent
strength so that the
selectivity can be
assessed in the first
place. The TLC on the
left was developed
in dichloromethane,
and the TLC on the
right in hexane/
dichloromethane 3:1.

Adding hexane reduces the solvent strength,

but does not affect the selectivity!

Figure 6:
Evaluation of the opti-
mal selectivity. In this
example this is clearly
in the system of selec-
tivity group VI, where
the individual compon-
ents have been sepa-
rated most effectively.
If there is only interest
in component 1 (top
mark), the choice is V.

Ideal = the other directly adjacent substances are

separated as well as possible from the substance of
interest.

4.3 Solvent strength

Every solvent has its own characteristic strength (which used to
be known as its polarity). The higher the figure, the stronger the
solvent and the quicker substances are transported through the
chromatographic system. Rapid transport through the column does
however mean that there is less interaction between the stationa-
ry and the mobile phase, and that the separation is therefore not
as effective. It is thus very important to have the correct solvent
strength so as to achieve optimum separation results.
Evaluation of the chromatographic system by thin-layer chromatography (TLC) 19

Solvent Group Strength Table 2:

Strengths of the most
n-Hexane – 0.1
common solvents.
Cyclohexane – 0.2
Diisopropyl ether I 2.4
Diethyl ether I 2.8
Ethanol II 4.3
Methanol II 5.1
Tetrahydrofuran III 4.0
Acetic acid IV 6.0
Dichloromethane V 3.1
Ethyl acetate VI 4.4
Aceton VI 5.1
Acetonitrile VI 5.8
Toluene VII 2.4
Xylene VII 2.5
Chloroform VIII 4.1

Using this table, solvents with different selectivity and usually

with different strengths can be set at identical solvent strengths and
thereby directly compared by mixing them with unselective solvents
such as hexane. The diagram in figure 8 shows the mixing ratios for
the most common solvents.

To successfully transfer TLC results to the flash cartridge, the

B = Solvent with higher polarity

Figure 7:
4.5 1 Setting the solvent
2 strength.
4.0 3 1 = Ethyl acetate
2 = Chloroform
3.5 3 = Tetrahydrofuran
4
4 = Dichloromethane
3.0 5 5 = Diisopropyl ether
Solvent strength Si

2.5

2.0

1.5

1.0

0.5

0.0
0 10 20 30 40 50 60 70 80 90 100
%B
20 Part 1 Flash Guide – Basics

solvent strength should be set so that the resulting substances are

approximately 0.15 – 0.4.

Figure 8:
Optimum Rf range
to transfer the results
to the flash cartridge
are 0.15 – 0.4.

Why such low Rf values? The reason for this is evident if we look
at the relationship between Rf values and column volume (CV).
An Rf value of 1 in the TLC means that the corresponding sub-
stance with the solvent front is flowing. The substance would also
move with the solvent front in a flash cartridge and after 1 column
volume would leave the cartridge. At an Rf value of 0.1 the flow
distance is 1⁄10 of the front distance – the substance would need
10 times longer to reach the front or in turn to reach the column
exit, i.e. 10 column volumes. The substance would be held back for
much longer and other components would therefore be separated.
The following applies to the relationship between column volume
and the Rf value:
– Column volume CV = 1⁄Rf
– Rf value ranging from 0.15 – 0.4, corresponding to 2.5 – 6.6
column volume.

Table 3: Rf value Column Rf value Column Rf value Column

Correlation of Rf volume CV volume CV volume CV
values and column 0.9 1.11 0.6 1.67 0.3 3.33
volumes.
0.8 1.25 0.5 2.00 0.2 5.00
0.7 1.43 0.4 2.50 0.1 10.0

Summary
Optimize the TLC conditions by applying the following rules:
1. Use identical silica gels if at all possible (same type and pore size)
for TLC plates and flash cartridges. Different silica gels behave
differently.
2. Look for suitable selectivity. The ideal selectivity separates the
components of interest well before adjacent components or im-
purities. The greater the difference, the more efficient the flash
separation.
3. Optimize the solvent strength. Ideal solvent strengths display
Evaluation of the chromatographic system by thin-layer chromatography (TLC) 21

Rf values ranging from 0.15 – 0.4 in TLC for the components of

interest; the ΔCV > 1.
Apply these conditions to the flash cartridge.

Figure 9

Example of pre-elution using TLC and transferring the results

to a Büchi cartridge

Step 1: Selectivity
Substance TLC 1 TLC 2 TLC 3
Rf CV ΔCV Rf CV ΔCV Rf CV ΔCV

1 0.54 1.8 0.56 1.8 0.61 1.6

0.5 0.6 0.3
2 0.43 2.3 0.42 2.4 0.45 1.9
0.6 1.4 1.0
3 0.34 2.9 0.27 3.8 0.35 2.9
3.3 8.7 2.3
4 0.16 6.2 0.08 12.5 0.19 5.2

Figure 10:
Evaluation of the
mobile phases in terms
of selectivity and
assessment.
1 = ethyl acetate
2 = diisopropyl ether
3 = chloroform
4 = dichloromethane

TLC 2 clearly displays

the best selectivity.
TLC 4 was not evalu-
ated.
22 Part 1 Flash Guide – Basics

Step 2: Solvent strength

Figure 11: Substance TLC

Setting the solvent Rf CV ΔCV
strength. The ratio of
hexane/diisopropyl 1 0.37 2.7
ether = 3:1. 1.3
Components 2 and 3
are of interest. 2 0.25 4.0
4.4
3 0.12 8.4
16.8
4 0.04 25.2

Step 3: Applying result to the cartridge

Figure 12:
Applying the conditions
to a Büchi flash
cartridge 12 x150 mm.
Eluent = hexane/
diisopropyl ether 3:1,
flow rate 14 ml/min,
detection = UV 254 nm.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (min)
 23

5 Injection/Column loading

Injecting the sample is usually a simple procedure in analytical chro-

matography. The quantities to be injected are low and solubility is
hardly an issue.
In preparative separations, on the other hand, the columns are
overloaded and the injection of the sample is of primary impor-
tance.
When loading the column the mixture to be separated should be
applied to the column bed in as compact a form as possible, i.e.
in a narrow horizontal band. Preparative separations are usually in
larger quantities, i.e. grams.
For a long time the general rule for preparative separations was
that a column can be loaded with an approximately 1% mixture,
in terms of the silica gel level. The use of modern flash systems
and optimizing the mobile phase (Rf 0.04 – 0.4, CV > 1) means that
nowadays the load can be increased to up to 10% – separation is
faster and more cost effective – more efficient all round!

Approximate possible load in g* Table 4:

ΔVS Cartridge Cartridge Cartridge Cartridge Approximate values
12x75 mm 12 x150 mm 40x75 mm 40x150 mm for loading at
Rf = 0.15 – 0.4.
1 0.15 0.3 1.2 2
2 0.3 0.6 2.5 5
6 0.6 1.2 5 10

* Values are given as a guide and depend on the silica gel used and the percentile
sample composition

Example of preparative separation at high load

Optimizing the conditions on the TLC plate silica gel 60:
Hexane/diisopropyl ether 95:5 (CV > 1, ΔCV > 1).
Substance Rf CV ΔCV Figure 13:
Optimized conditions
1 0.95 1.05
on TLC.
1.1
2 0.48 2.1
2.2
3 0.23 4.3
24
0 2 4 6 8 10 12 14 16 18 20
Time (min)
Figure 14:
The impact of incre-
asing the load on the
separation. Cartridge
12 x150 mm. Silica gel
60, 40 – 63 µm, eluent
hexane/diisopropyl
ether 95:5, 14 ml/min,
load (from left to right)
300 mg, 600 mg
and 1200 mg.
Injection volume 1 ml.
Table 5
Part 1 Flash Guide – Basics
0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20
Time (min) Time (min)
The volume must be low so that the sample can be compactly
applied to the column bed. If the volume is too high, the band is
considerably widened and the separation is less efficient.
The sample that is to be separated can be brought into the se-
paration system either as a solution or dry, adsorbed by silica gel.
A classical solution sample injection requires that the sample can
be sufficiently dissolved in the starting eluent. The injection volume
should be no more than 10% of the column volume. The following
injection volumes apply to the Büchi cartridges:
Recommended max. injection volumes
Cartridge Cartridge Cartridge Cartridge
12x75 mm 12 x150 mm 40x75 mm 40x150 mm
1.5 ml 2 ml 10 ml 20 ml
If the sample cannot be sufficiently dissolved in the starting elu-
ent, dry application can be carried out, where the sample is dissol-
ved in any solvent and mixed with silica gel. The solvent is then
distilled off using rotation. This dry silica gel is packed into a preco-
lumn and this is then fitted in front of the separation column into the
eluent flow. The components to be separated are then constantly
eluted from the precolumn to the actual separation column. This
procedure is also advisable if there are sticky or solid impurities in
the sample which cannot easily be removed!
Another slightly unconventional injection method for samples in
the form of solutions is not to dissolve the sample in the starting
eluent but in a completely different solvent with excellent dissolving
properties for the mixture. This “foreign” solvent is separated in the
separation flow as an additional component. Retention times are
usually in the range of the solvent front. If the components of inte-
rest are optimized to an Rf range of 0.15 – 0.4, separating the front
is not a problem anyway.
Injection /Column loading 25

Toluene

0 2 4 6 8 10 12 14 16
Time (min)

Figure 15:
This simple and unconventional injection procedure is often used Liquid sample injection
to inject by-products that are not easily dissolved to the separati- in toluol, eluent = he-
on column. These substances are usually heavily adsorbed in the xane/diisopropyl ether
9:1.
area where they initially enter the column and remain there. This is
not, however, a problem if disposable cartridges are used, as the
cartridge is changed anyway after the components of interest have
been eluted.

Special advice for injecting dissolved samples

In preparative chromatography, it is often not possible to spend
the time pre-cleaning the samples and the mixtures are applied
directly to the separation column with varying levels of accom-
panying substances. To ensure that the flash equipment operates
smoothly it is therefore very important to rinse the injection port
clean after every injection, regardless of whether it is a quick
stop valve or a device fitted with a tap system. This is the only
way to avoid problems such as sample contamination or leaking
injection ports. The following should therefore be observed in the
injection process:

1. Stop pump
2. Inject sample
3. Rinse injection port
4. Start pump
26 Part 1 Flash Guide – Basics

6 Gradient elution

The examples given are all separated isocratically, i.e. the mobi-
le phase is identical throughout the entire separation process. In
practice this is, however, often not possible, as the substances to
be separated in adsorption often differ.
Substances that cannot be successfully eluted isocratically can
be identified by pre-elution using TLC, and can be optimised accor-
dingly. The following example explains the procedure:

a) Establishing the suitable selectivity (see 4.2).

Figure 16:
Suitable selectivity b) Establishing the suitable solvent strength
using ethyl acetate.

This is problematic – either the Rf values are so high that it is practi-

cally impossible to achieve separation with a preparative loading of
the column, or the Rf values are so low that they can only be eluted
from the column with a great deal of time and solvent.

Figure 17:
Different solvent
strengths, achieved
using hexane with
varying levels of ethyl
acetate.
Gradient elution 27

As there is no basic common denominator for the conditions, the

Rf values for highly adsorbent and less adsorbent components are
optimised separately (both Rf values from 0.15 – 0.04).

Figure 18:
Selection the mobile
phase for a step
elution hexane with
varying levels of ethyl
acetate according
to the indication on
the TLC plate.

These are the conditions that apply when applying the separa-
tion to the column. The less adsorbent components are eluted
with the weaker eluent. We then switch to the polar mobile phase
(= higher level B). Depending on the equipment used, this switch
can either be carried out gradually or in one step.
The following examples show how the separation can be affec-
ted by the choice of solvent strength. The level of ethyl acetate is
entered in the chromatogram (% B).

Figure 19:
100 Separation using a
step gradient
10% B / 25% B.
80

60
%

40

20

0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (min)

Absorption %B
28 Part 1 Flash Guide – Basics

Figure 20:
Separation with a 100
continuous gradient
10 ⇒ 20% B in 10 min,
80
20 ⇒ 45% B in 5 min,
then 45% B constant.
60

%
40

20

0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (min)

Absorption %B

Figure 21:
Separation with 100
continuous gradient
10% B for 9 min,
10 ⇒ 45% B in 8 min, 80
then 45% B constant.

60
%

40

20

0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (min)

Absorption %B
126 Appendix

6 Index

absorption 17, 54, 55, 114

adsorbent 14, 27, 40, 62, 74, 80, 115
adsorption chromatography 34, 40, 81
affinity chromatography 39, 51
agarose 39, 44
alumina 42, 53
anion exchange 39
appropriate column 88
boiling point 46, 51, 119
Büchi Cartridger C-670 92
capacity factor k’ 66, 72, 83
cartridge 20, 92
cation exchange 39
charge-transfer complex 35
chemisorption 42
chromatogram 58, 105, 120
cleaning of columns 102
column 12, 23, 24, 58, 74 ff, 88, 105
column efficiency 74 ff
column length 61, 67, 77, 78, 79, 116
column packing 58 ff, 89 ff, 91
column test 97
columns in series 79
π-complex 35
conditioning 89, 96
conductivity detector 56
dead volume 37, 63, 123
delivery 77, 116
detection 17, 48, 54
dextran 44, 45
dipole 34, 48, 50, 51, 66, 67, 68
dipole interactions 34, 35
dry packing method 90
eluent 50, 66, 87, 119
eluotropic serie 49 ff, 51, 119, 121
elution 26, 42, 43
elution sequence 15, 42, 43
elution time 47, 49
equipment 104
extinction 54, 55, 114, 121
flow rate 61, 74, 76, 77, 115, 116
formulae 110
fraction collector 104, 105
fraktogel 45
fronting 121
gel chromatography 36, 44, 51, 94, 119
GFC 44, 45, 101
Glatz 63
GPC 44, 45
Halász 74
Helmchen 63
Hildebrand 47, 70
Index 127

hydrogen bridge bonds 35

increase factor 78, 79, 116
injection 23 ff, 104
interpretation of TLC 82
ion-exchange chromatography 39
ionic strength 52
isocratic chromatography 121
linear flow rate 61, 74 ff, 112, 115, 116
loading 23, 80
miscibility 46, 117
mobile phase 16 ff, 27, 46 ff, 61, 66 ff, 85 ff, 121
mobile phase reservoir 104
net retention time 112, 121
normal phase silica 40, 98
number of theoretical plates 60 ff, 73, 76 ff, 84, 110, 114, 115, 120
optimum plate height 75
packing method for soft and rigid gels 94
particle diameter 42, 62, 74
particle size 40, 41, 42, 61, 62, 74 ff, 115
peak width at half height 59, 122
permeation volume 38
plate height 61, 74 ff, 115
polarity 17 ff, 47 ff, 66 ff, 122
polyacrylamide 44
polyamides 43
pore 36 ff, 41, 61
proton (H)-acceptor 48, 50, 66 ff, 117, 119
proton (H)-donor 48, 50, 66 ff, 117, 119
pump 104
purity 32, 49, 64
reduced plate height 62
refractive index 51, 55, 119
refractive index detector 55
relative retention 67
resolution 62, 64, 72, 77 ff, 84, 111, 116, 120
retention factor Rf 82
retention time 59, 67, 112, 122
reversed phase 43, 67, 100, 122
reversed phase chromatography 43, 50, 51, 66
SEC 37
selectivity 16 ff, 42, 47 ff, 70 ff, 86, 118, 123
selectivity triangle 16, 48, 70, 85, 117
separation factor α 67, 72, 73, 83, 114
separation mechanisms 34 ff, 81
sephadex 44, 95
sepharose 45
silica gels 35, 40 ff
size exclusion chromatography 36 ff
slurry 93 ff, 123
slurry packing method 93
Snyder 17, 47, 48, 70, 85
solvent strength 16 ff, 47 ff, 70 ff, 118
stationary phase 15, 40 ff, 123
step gradient 27, 123
128 Appendix

steric effects 35
symmetry index 59, 60, 62, 113
tailing 123
test chromatogram 98, 99, 100, 101
test mixture 98, 100, 101
theoretical plate number 60
thin-layer chromatography 16, 81 ff
TLC optimization 83, 84
total permeation 37
transmittance 55, 114
UV absorption 54
UV detector 54, 55, 56
UV limit 17, 48, 50, 51, 70, 85, 118, 119
viscosity 51, 61, 119
BÜCHI Labortechnik AG
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Switzerland
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